1. Field of the Invention
The present invention generally relates to methods of detecting PML-containing NBs and proteins therein for cytodiagnostic staging of neoplasia and squamous cell carcinoma. Specifically, the present invention relates to methods of diagnosing the different stages of cervical neoplasia and squamous cell carcinoma.
2. Description of the Related Art
Cervical cancer is one of the leading causes of cancer-related death in women worldwide and is the leading cause of cancer-related death in women in developing countries. Worldwide, there are approximately 500,000 new cases per year of which 15–20,000 cervical cancers are diagnosed in the United States. Moreover, abnormalities of the precancerous disease of the cervix occur in an estimated 500,000 to 1,000,000 women each year in the United States.
Cervical squamous cell carcinomas originate from a multilayered cervical epithelium and develop progressively over the course of years. See DiSaia, P J and Creasman, W T (1993) CLINICAL GYNECOLOGIC ONCOLOGY, 4th ed. Mosby Year Book pp. 1–36; and Singer, A (1976) THE CERVIX London: The Whitefriars Press Ltd. pp. 87–104. The tissue goes through a spectrum of hyperplastic changes classified clinically as cervical intraepithelial neoplasia (CIN), ranging from CIN I (mild dysplasia), followed by CIN II (moderate dysplasia) to CIN III (severe dysplasia), where it may eventually result in invasive cervical cancer. The molecular mechanisms that regulate the induction and progression of cervical squamous cell carcinoma (SCC) remain an unsolved problem of great clinical relevance. The main risk factors for cervical cancer include human papilloma virus infection, multiple sexual partners, long-term cigarette smoking, immunosuppression, and the use of oral contraceptive. See Brisson, J, et al. (1994) America Journal Epidemiology 140:700–710; Lowy, D R, et al. (1994) PNAS USA 91:2436–2440; and Zur Hausen, H (1991) Virology 184:9–13.
The promyelocyte (PML) protein was identified first in the pathogenesis of acute promyelocytic leukemia (APL). See Lin, R J, et al. (1999) Trends In Genetics 15:179–183; and Ruggero D, et al. (2000) Bioessays 22:827–835. This type of leukemia is categorically characterized by a chromosomal translocation t(15;17), which fuses the PML gene to the retinoic acid receptor α (RARα). See de Thé, H, et al. (1990) Nature 347:558–561; Borrow, J, et al. (1990) Science 249:1577–1580; Longo, L, et al. (1990) J Exp Med 172:571–575; Kakizuka, A, et al. (1991) Cell 66:663–674; de Thé, H, et al. (1991) Cell 66:675–684; Goddard, A D, et al. (1991) Science 254:1371–1374; Pandolfi, P P, et al. (1991) Oncogene 6:1285–1292; Weis, K, et al. (1994) Cell 76:345–356; and Ruggero, D, et al. (2000) Bioessays 22:827–835. In normal cells, PML is found in discrete nuclear structures known as nuclear bodies (NBs) or PML oncogenic domains (PODs). See Dyck, J A, et al. (1994) Cell 76:333–343; Koken, M H, et al. (1994) EMBO J. 13:1073–1083; and Weis, K, et al. Cell 76:345–356. In APL cells, PML is displaced and presents in a microspeckled pattern. This altered pattern is thought to cause the disruption of the normal function of PML. See Lin, R J, et al. (1999) Trends In Genetics 15:179–183.
While a host of studies exist in the literature, a defined function has yet to be assigned to PML and its corresponding nuclear body. See Ruggero, D, et al. (2000) Bioessays 22:827–835. Studies on the biologic function of PML demonstrate that PML acts in a variety of cellular processes including apoptosis, cell cycle progression, and transcriptional regulation. See Mu, Z M, et al. (1994) Molecular and Cellular Biology 14:6858–6867; Le, X F et al. (1996) J. Biol. Chem. 271:130–135; He, D, et al. (1997) Cancer Res. 57:1868–1872; Le, X F, et al. (1998) Oncogene 16:1839–1849; Mu, Z M, et al. (1997) Carcinogenesis 18:2063–2069; Doucas, V, et al. (1993) PNAS USA 90:9345–9349; and Vallian, S, et al. (1998) Oncogene 16:2843–2853.
PML expression is modulated by multiple stimuli including viral infection, γ-irradiation, estrogen, and interferon. See Ruggero, D, et al. (2000) Bioessays 22:827–835; Maul, G G, et al. (1993) J. Gen. Virol. 74:2679–2690; Maul, G G, et al. (1994) J. Gen. Virol. 75:1223–1233; Lavau, C, et al. (1995) Oncogene 11:871–876; Mueller, S and Dejean, A (1999) J. Virology 73:5137–5143.
PML is overexpressed in distinct pathological situations that are associated with stimulated transcription and cell hyperactivity, such as in inflammatory and tumorous states. See Lavau, C, et al. (1995) Oncogene 11:871–876; Gambacorta, M, et al. (1996) Am. J. Pathology 149:2023–2035; and Maul, G G, et al. (1995) J. Cellular Biochem. 59:498–513. While the amount of PML is low in normal tissues, an increase in PML expression is observed immumohistochemically in tumors of various origins. See Koken, M H, et al. (1995) Oncogene 10:1315–1324.
Further, it was found that PML is covalently modified by a small polypeptide called SUMO-1, which triggers targeting of PML to its corresponding NBs. See Sternsdorf, T, et al. (1997) J. Cell Bio. 139:1621–1634; Duprez, E, et al. (1999) J. Cell Science 112:381–393; Kretz-Remy, C and Tanguay R M (1999) Biochem. Cell Bio. 77:299–309; and Zhong, S, et al. (2000) Blood 95:2748–2752. SUMO-1 is an ubiquitin-like protein with significant sequence homology to ubiquitin. See Hodges, M, et al. (1998) Current Bio. 8:749–752. Several substrates for SUMO-1 have been reported. See Zhong, S, et al. (2000) blood 95:2748–2752; Desterro, J M, et al. (1998) Mol. Cell Bio. 2:233–239; Saitoh, H, et al. (1998) Current Bio. 8:121–124; Gostissa, M, et al. (1999) EMBO J. 18:6462–6471; and Rodriguez, M S, et al. (1999) EMBO J. 18:6455–6461.
SUMOylation plays a role in multiple vital cellular processes such as oncogenesis, cell cycle control, apoptosis and response to viral infection. Conjugation to SUMO-1 is thought to be a prerequisite for PML to maintain the nuclear body and for subsequent localization of other protein components of the body. In contrast, it has been shown that SUMO-1 modification of PML is not necessary for PML to target the NB. See Ishov A M, et al. (1999) J. Cell Bio. 147:221–233. PML is degraded in cells infected by several viruses. See Everett, R D, et al. (1994) EMBO J. 13:5062–5069; Anh, J H, et al. (1998) Mol. Cell Bio. 18:4899–4913; and Ahn, J H, et al. (1999) J. Virology 73:10458–10471. Infection by a number of DNA viruses triggers the reorganization of the NBs, suggesting an important role for the NBs in the viral infection process. The ability of viral proteins to specifically abrogate the covalent SUMO-1 modification of PML and SP100 is directly linked to their capacity to disassemble NBs. The biological significance of the destruction of the body in viral infection is still unclear.
Immunocytochemical studies to assist in the diagnosis of the progression or extent of the disease are prevalent in the clinical setting, as is the case of breast cancer. To date, while some markers for cervical neoplasia have been reported, their expression is not a footprint for a particular stage of the disease. See Litvinov, S V, et al. (1996) American Journal of Pathology 148:865–875; Skyldberg, B, et al. (1999) J. Nat'l Cancer Inst. 91:1882–1887; Vassallo, J, et al. (2000) International Journal of Gynaecology and Obstetrics 71:45–48; and Klaes, R, et al. (2001) Internat'l J. of Cancer 92:276–284. Immunohistochemical detection of PML expression as a marker for disease progression has focused mainly on hematopoietic malignancies, particularly APL. Although reports on PML in other solid tumor cancers have been documented, neither systematic mapping of the changes in PML throughout the progression of the cancer from CIN to SCC has been performed; nor, has a correlation to its colocalization with SUMO-1. See Saitoh, H, et al. (1998) Current Biol. 8:121–124; and Terris, B, et al. (1995) Cancer Res. 55:1590–1597. These studies also relied on immunohistochemical findings that are graded on the presence or absence of a colorimetric substrate in the nucleus. No spatial or quantitative information at the molecular level of the PML-containing NBs can be acquired from these data.
Thus, a need still exists for methods of detecting PML-containing NBs and methods of diagnosing the different stages of cervical neoplasia and squamous cell carcinoma.